Mutually Exclusive Signaling Signatures Define the Hepatic and Pancreatic Progenitor Cell Lineage Divergence

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RESOURCE/METHODOLOGY

Mutually exclusive signaling signatures define the hepatic and pancreatic progenitor cell lineage divergence

Elisa Rodrı´guez-Seguel,1 Nancy Mah,2 Heike Naumann,1 Igor M. Pongrac,1 Nuria Cerda´-Esteban,1 Jean-Fred Fontaine,2 Yongbo Wang,3 Wei Chen,3 Miguel A. Andrade-Navarro,2 and Francesca M. Spagnoli1,4 1Laboratory of Molecular and Cellular Basis of Embryonic Development, 2Computational Biology and Data Mining, 3Laboratory for Novel Sequencing Technology, Functional and Medical Genomics, Max Delbru¨ ck Center for Molecular Medicine, D-13092 Berlin, Germany

Understanding how distinct cell types arise from multipotent progenitor cells is a major quest in stem cell biology. The liver and pancreas share many aspects of their early development and possibly originate from a common progenitor. However, how liver and pancreas cells diverge from a common endoderm progenitor population and adopt specific fates remains elusive. Using RNA sequencing (RNA-seq), we defined the molecular identity of liver and pancreas progenitors that were isolated from the mouse embryo at two time points, spanning the period when the lineage decision is made. The integration of temporal and spatial gene expression profiles unveiled mutually exclusive signaling signatures in hepatic and pancreatic progenitors. Importantly, we identified the noncanonical Wnt pathway as a potential developmental regulator of this fate decision and capable of inducing the pancreas program in endoderm and liver cells. Our study offers an unprecedented view of gene expression programs in liver and pancreas progenitors and forms the basis for formulating lineage-reprogramming strategies to convert adult hepatic cells into pancreatic cells. [Keywords: liver; pancreas; mouse; RNA-seq; Wnt signaling] Supplemental material is available for this article. Received April 18, 2013; revised version accepted July 29, 2013.

The liver and pancreas share many common aspects of The liver originates solely from the ventral foregut their early embryonic development as well as some adult endoderm, adjacent to where the ventral pancreas (vpa) functional properties and both are endowed with essen- emerges, and the two cell fates are specified concomi- tial metabolic functions (Slack 2007; Zaret 2008). This tantly by mouse embryonic day 8.5 (E8.5) (Deutsch et al. close relationship makes the liver a very attractive tissue 2001). Previous studies showed that the ventral foregut source for generating new pancreatic b cells by lineage endoderm is multipotent for the hepatic and pancreatic reprogramming, which might be used in the context of programs (Deutsch et al. 2001; Tremblay and Zaret 2005; cell-based therapy of diabetes. However, how liver and Miki et al. 2012). Hepatic and pancreatic endoderm ex- pancreas cells diverge from a common endoderm progen- press a common set of transcription factors, such as the itor population and adopt specific fates remains elusive. FoxA and GATA factors, and are exposed to the same During embryonic development, the pancreas orig- extrinsic signals: fibroblast growth factor (FGF) and BMP inates from distinct outgrowths of the dorsal and ventral (Deutsch et al. 2001; Chung et al. 2008; Spagnoli and regions of the foregut endoderm (Spagnoli 2007; Puri and Brivanlou 2008; Zaret 2008). However, the develop- Hebrok 2010). Subsequently, the two buds fuse to form mental regulators of the fate choice between liver and a single organ containing both pancreatic exocrine and pancreas are poorly understood. It is also unknown how endocrine cells (Spagnoli 2007; Puri and Hebrok 2010). the pancreatic program is stably repressed in nascent hepatic progenitors and how the hepatic program is repressed in pancreatic progenitors. This knowledge will 4Corresponding author E-mail [email protected] aid in not only the programming of stem cells to pancre- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.220244.113. atic and hepatic lineages, but also the discovery of the

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RNA-seq of hepatic and pancreatic progenitor cells mechanisms underlying cellular plasticity between liver foregut endoderm of Tg(Prox1-EGFP) embryos, the local- and pancreas. ization of EGFP perfectly mirrored endogenous Prox1 Studies conducted on various stem cell types or whole expression and overlapped with other endodermal genes, organisms (Tumbar et al. 2004; Wang et al. 2004; Deque´ant such as Foxa2, at E8.5 and with tissue-specific genes, such et al. 2006; Mitiku and Baker 2007) have clearly demon- as Pdx1 in the pancreas or Liv2 in the liver, from E9.5 strated that significant insights into the regulation of the onward (Fig. 1A,B; Watanabe et al. 2002; Lee et al. 2005; cell fate decision are obtained through global analysis of Wandzioch and Zaret 2009; Puri and Hebrok 2010). Thus, gene expression events. Thus, to search for developmen- the Tg(Prox1-EGFP) in vivo model enabled us to visualize tal regulators that are involved in the control of the hepatic and pancreatic progenitors under fluorescence pancreas and liver cell fate decision, it is fundamental microscopy before organogenesis had started. Distinct to have a global picture of the regulation of gene expression regions of the prospective hepatic and pancreatic endo- in the mammalian hepatic–pancreatic endoderm lineage. derm were manually microdissected, and Prox1-EGFP+ To this aim, we performed sequencing-based expression cells were FACS-purified and subjected to RNA-seq profiling (RNA sequencing [RNA-seq]) on hepatic and analysis to define their transcriptomes (Fig. 1C–E). Pre- pancreatic progenitors in mice at distinct developmental vious fate mapping studies in mouse and chick embryos stages. We devised a strategy to purify liver and pancreas suggested differential locations of hepatic and pancreatic progenitor populations directly from the endoderm of progenitors in the ventral foregut at the early somite stage Tg(Prox1-EGFP [enhanced green fluorescent protein]) (Tremblay and Zaret 2005; Miki et al. 2012). To determine reporter mouse embryos before and after the onset of whether regional identity within the foregut is associated organogenesis. By integrating the temporal and spatial with differential gene expression, we collected Prox1- gene expression profiles, we found mutually exclusive EGFP+ cells of the whole ventral foregut (referred to as fg) signaling signatures in hepatic and pancreatic progeni- and exclusively of the medial ventral foregut (referred to tors. Importantly, we identified the noncanonical Wnt as mfg) from E8.5 embryos at the same somite stage pathway as a potential developmental regulator of the (seven to nine somites) (Fig. 1C, top). At E10.5, budding pancreas versus liver fate decision, since it is expressed in sites of the liver and pancreas, including both the dorsal the foregut endoderm before the cell fate choice is made pancreas (dpa) and vpa, were readily distinguishable, and and then is maintained in pancreas progenitors but is the three distinct progenitor populations were isolated absent in hepatic progenitors. Moreover, when assayed in and processed for the analysis (Fig. 1C, bottom). Xenopus embryos and mammalian cells, activation of the Given the small number of progenitor cells, we applied noncanonical Wnt pathway is able to promote pancreatic a submicrogram RNA-seq method and used paired-end fate, suggesting an ancient mechanism for controlling the sequencing technology (Adamidi et al. 2011). A compa- pancreas versus liver fate choice. Overall, our study offers rable number of high-quality raw reads was obtained from an unprecedented view of gene expression programs in each sample and used to estimate the relative abundance liver and pancreas progenitors during the defined period of of transcripts (Table 1; Trapnell et al. 2010). Principal their lineage divergence and provides novel insights into component analysis (PCA) on the nonredundant gene key mechanisms that may underpin cellular plasticity and expression data showed that individual samples perfectly reprogramming between the two cell types. clustered according to their embryonic stage and/or region of origin (Fig. 2A). Known hepatic and pancreatic tissue-specific transcripts were found in the E10.5 liver Results and pancreas RNA-seq profiles, respectively, reflecting the high reliability and accuracy of the data obtained RNA-seq on FACS-purified hepatic and pancreatic (Fig. 1E; Supplemental Fig. 1). progenitors To explore the transcription program associated with Integrating temporal and spatial transcription profiles liver and pancreas progenitors in vivo at the time of their of the progenitor populations fate divergence, we performed RNA-seq on distinct mouse endoderm progenitor populations isolated at two To identify developmental regulators of pancreatic and/or developmental stages. For in vivo monitoring of hepatic hepatic fate, we integrated the temporal and spatial and pancreatic progenitor cells, we used the transgenic analyses. Both hepatic and pancreatic progenitors arise mouse line Tg(Prox1-EGFP)Gsat/Mmcd that carries the re- from foregut endoderm cells (Spagnoli 2007; Zaret 2008). porter gene EGFP into the Prox1 homeobox gene locus Therefore, the set of genes expressed in the foregut (fg (Gong et al. 2003). Prox1 is the earliest specific marker in or mfg) is expected to include regulators of both early common between hepatic and pancreatic endoderm from pancreatic and hepatic fate specification as well as the gastrulation onward (Burke and Oliver 2002; Wandzioch choice between the two fates (liver and pancreas). We and Zaret 2009) and therefore is ideally suited for iso- compared those genes with the genes expressed later in lating both hepatic and pancreatic progenitors (Fig. 1). the E10.5 liver, vpa, and dpa and found a number of genes We showed that Prox1-EGFP transgenic mouse embryos specific to either liver or pancreas that were already reproduced the endogenous pattern of Prox1 expression expressed at the earlier time point in the E8.5 foregut in the endoderm from E7.5 onward (Fig. 1A,B; Burke and (89 and 517 genes, respectively) (Fig. 2B, left). Of interest, Oliver 2002; data not shown). In both ventral and dorsal these numbers suggest a higher divergence between the

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Rodrı´guez-Seguel et al.

Figure 1. In vivo isolation and RNA-seq profiling of endoderm progenitor cells from Tg(Prox1-EGFP) mouse embryos. (A) Rep- resentative maximum confocal Z-projections of immunofluorescence analysis in Tg(Prox1-EGFP) E8.5 mouse embryos shows EGFP reporter expression in both ventral (arrows) and dorsal (asterisk) foregut cells, mirroring endogenous Prox1 expression (see the inset for overlay) and overlapping with Foxa2 expression domains at the three-somite (3S) stage. Embryos are presented in ventral view. (B) In Tg(Prox1-EGFP) E9.5 mouse embryos, EGFP expression was detected in the hepatic and both dorsal and ventral pancreatic buds, mirroring the endogenous Prox1 (see the inset for overlay). EGFP colocalized with Pdx1 in both pancreatic buds and with Liv2 solely in the liver bud. Embryos are presented in lateral view. (C–E) Schematic representation of cell sampling and the RNA-seq procedure. (C)EGFP+ fg and mfg endoderm at the seven- to nine-somite (7–9S) stage/E8.5 and liver (lv), ventral (vpa), and dorsal (dpa) pancreas at E10.5 were microdissected from Tg(Prox1-EGFP) embryos. Cells were dissociated and subjected to FACS. (D) Representative diagram of the EGFP+ cell fraction isolated by FACS. The dashed box indicates EGFP+-gated cells, and cells negative for EGFP are in purple. Transcript expression was profiled by RNA-seq. (E) Example illustrating the RNA-seq read coverage profile of the pancreatic-specific gene Pdx1.TheY-axis indicates the number of read counts in each cell population (Y-axis scale is 0 1000 counts). FPKM (fragments per kilobase of exon per million fragments mapped) values for Pdx1 in each data set are included on the right. Exons are depicted as gray boxes at the bottom on the X-axis. As expected, a large number of reads was found in pancreatic progenitors. Bars: A, 100 mm; B,50mm. foregut and liver when compared with the number of (211 vs. 257) (Fig. 2B, right) indicates a large divergence genes whose expression is instead maintained in the between the two buds, reflecting the dual embryological pancreas (vpa and dpa) (Fig. 2B, left; Supplemental Fig. origin of the pancreas. 2). This is in line with the hypothesis of the pancreas being the default fate of the ventral foregut endoderm Different Wnt signaling signatures correlate (Deutsch et al. 2001). In contrast, genes initially showing with pancreatic or hepatic progenitor states low expression in the foregut but high expression later in E10.5 hepatic or pancreatic progenitors represent cell To study the mechanisms underlying the pancreas versus type-specific markers of differentiation and possibly fac- liver fate decision, we focused on the transcripts whose tors involved in late aspects of organogenesis (Fig. 2B, expression was detected in the E8.5 foregut and main- right; Supplemental Fig. 3). At this later time point, the tained exclusively in the pancreatic rudiments and there- number of genes that are unique to either the dpa or vpa fore possibly was endowed with a role in this cell fate

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RNA-seq of hepatic and pancreatic progenitor cells

Table 1. Summary of sequencing data and annotation (Fig.2D).Theseresultssuggest a cell type-dependent non- information canonical Wnt activation in the foregut and pancreatic Number of transcriptsa progenitors that is ensured by not only distinct classes of ligands, but also recruitment of unrelated coreceptors, as Sample Raw reads Mapped reads Annotated Unannotated previously described in other contexts (Grumolato et al. fg 121,497,737 115,767,182 38,159 19,348 2010). For instance, the differential expression of Ror2 mfg 114,786,211 108,295,227 38,254 19,347 coreceptor might sustain noncanonical Wnt activation in Liver 114,436,606 109,182,827 38,260 19,363 foregut and pancreas progenitors but not in liver progenitors. vpa 109,534,481 102,391,351 38,171 19,371 dpa 104,526,851 99,464,506 38,168 19,356 The transcript abundance was calculated by estimating the frag- Noncanonical Wnt signaling signature underlies ments per kilobase of exon per million mapped fragments (FPKM). the pancreas versus liver fate decision An average of 38,000 annotated distinct transcripts per sample was If multiple components of a pathway are expressed and detected. the expression is developmentally regulated, it is very aThis includes all Cufflinks genes found in each sample before filtering. Annotated transcripts were considered to be all Cufflinks likely that the pathway is active. Therefore, we under- genes that were associated to a gene symbol. took several approaches to study the possible role of the Wnt noncanonical pathway in the process of pancreas versus liver cell fate specification. For further analysis, restriction. We hereafter refer to this cluster as FP (foregut we prioritized FP group transcripts that showed signifi- pancreas) (Fig. 2B, left). A gene ontology (GO) term analysis cant differential regulation between the pancreatic and of our results revealed significant enrichment for Wnt- hepatic endoderm (150 transcripts out of 517; Cufflinks, related ‘‘Molecular Function’’ categories in the group FP, P-value < 0.05) (Figs. 2D, 3A). We performed quantitative such as Wnt protein-binding (GO:0017147; seven genes; RT-qPCR and immunofluorescence staining to confirm P-value < 0.00001.9) and Wnt-activated receptor (GO: that the transcripts identified as differentially expressed 0042813; five genes; P-value < 0.00035) activities. Among by RNA-seq indeed showed significant enrichment in the the Wnt-associated factors were genes such as Frizzled 2 pancreatic cell lineage (Fig. 3B,C; Supplemental Fig. 5). In (Fzd2), Fzd4, Ror2, Sfrp5, Sfrp2,andWls (Supplemental Drosophila as well as some mammalian tissues, the PCP Table 1). Also, Wnt-related ‘‘Biological Process’’ categories proteins are initially enriched in the apical cell mem- were primarily enriched in the group FP, such as estab- brane prior to their asymmetric distribution at either the lishment of epithelial cell polarity (GO: 0090162), Wnt proximal or distal side of the cell (Vichas and Zallen 2011; receptor signaling pathway (GO: 0007223), and estab- Wallingford 2012). Their distribution within the plane of lishment of planar polarity (GO: 0001736) (Supplemental the foregut epithelium has not been previously reported. Table 2). Of interest, no other regions of the Venn diagram We found Fzd2 receptor, Ror2 coreceptor, and PCP pro- displayed such a significant enrichment for Wnt-related teins such as Celsr2 and Fat1 to be enriched at the cell categories as the group FP, suggesting a unique signaling surface of both foregut epithelial and pancreatic progen- signature of the pancreas versus liver fate in the mam- itor cells with no obvious asymmetric distribution but malian endoderm. completely absent in hepatic progenitors (Fig. 3C; Sup- We decided to further characterize the expression dy- plemental Fig. 5A). namics of Wnt signal transducers. Interestingly, in our In several systems, there is direct evidence that non- data set, we found that intracellular transducers of the canonical Wnt signaling regulates cell adhesion and cy- canonical Wnt/b-catenin signaling (Grigoryan et al. 2008; toskeleton components (Karner et al. 2009). Accordingly, Clevers and Nusse 2012)—such as b-catenin (Ctnnb1), in our data set, several adhesion molecules—including APC, Axin, and TCFs—as well as the common canonical Claudin 4, laminin, integrin a3 (Itga3), fibronectin leu- and noncanonical component Dishevelled 1 (Dvl1)(Angers cine-rich transmembrane protein 2 (Flrt2), and Flrt3—as and Moon 2009; Kikuchi et al. 2009) were expressed at well as cytoskeleton components such as cytokeratins stable levels in the different progenitor populations CK19 and CK7 and Shroom3 showed similar differential (Fig. 2C), whereas transcripts encoding ligands, receptors, expression, and were down-regulated in hepatocyte pro- and coreceptors exhibited lineage-specific induction or genitors after lineage divergence (Fig. 3B,C; Supplemental repression (Fig. 2D). In particular, only noncanonical Wnt Fig. 5B). Taken together, these results indicate that active ligands (Wnt5a, Wnt5b, and Wnt7b) (Kikuchi et al. 2009; remodeling of the foregut epithelium accompanies cell Grumolato et al. 2010) were found in the foregut and fate determination between liver and pancreas, resulting pancreas and all of them were strongly down-regulated in in differences in the relative adhesiveness of the cells. liver progenitors (Supplemental Fig. 4). Additional de- Moreover, our findings suggest that the noncanonical terminants of noncanonical/planar cell polarity (PCP) Wnt signaling might influence this cell fate decision Wnt specificity displayed similar expression profiles, in- within the foregut endoderm. cluding the receptors Fzd2 and Fzd7 and the PCP core One prediction from this hypothesis is that upon ex- membrane proteins Stbm/Vangl2, Celsr2, and Fat1 (Fig. 2D). posure to noncanonical Wnt, endodermal cells acquire Finally, the coreceptors Lrp5 and Ror2 showed divergent a pancreatic-specific differentiation program and fail to expressions—one strongly induced in the liver, and the induce hepatic genes expression. To test this prediction, other one induced exclusively in pancreas progenitors we first used one of the most intensively studied

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Figure 2. Temporal and spatial integration analysis of the RNA-seq-derived transcriptome profiles. (A) PCA shows that the RNA-seq- derived transcriptome profiles are characteristic of different progenitor cell types (for detailed description, see the Materials and Methods). (B) Venn diagrams showing the number of unique and common highly expressed transcripts between progenitor cells at different developmental stages. To further focus our analysis on subsets of genes with distinct expression patterns, we divided the working data set into two groups based on a cutoff for high expression (defined as FPKM = 10, at approximately the 50th percentile of RNA-seq expression for each sample). Of 14,053 genes, 8110 could be categorized in the defined Venn regions. For example, 5437 genes exhibited relative abundance values of >10 FPKM in all samples, while 517 genes were highly expressed in the foregut, vpa, and dpa but not in the liver (referred to as group FP). In contrast, 89 genes were highly expressed in the foregut and liver but not in the vpa and dpa. Three-hundred-sixty-three transcripts were present (>10 FPKM) only in the fg but not in the liver, dpa, and vpa (not shown in the diagram). As shown in the PCA plot in A, fg and mfg were highly similar; therefore, these samples were combined together as ‘‘foregut’’ to simplify the visualization. (C,D) Levels of Wnt signaling pathway gene expression across the fg, liver (lv), vpa, and dpa progenitors. FPKM values (Y-axis) were plotted against the different progenitors cell types (X-axis). (*) Wnt factors present in the 150 FP group transcripts that showed significant differential regulation between the pancreas and liver. noncanonical Wnts, Wnt5a, which we also found to be et al. 2012). Moreover, the expression pattern appears cell-autonomously produced by the E8.5 foregut endoderm conserved across species because Wnt5a is expressed in (Supplemental Table 1; Supplemental Fig. 4A–C). Sub- endodermal as well as surrounding mesodermal cells in sequently, at E10.5, Wnt5a expression was maintained at the Xenopus embryo at the time of foregut organogenesis lower levels in pancreatic progenitor cells and was absent (Supplemental Fig. 4D; Zhang et al. 2013). Therefore, we in hepatoblasts (Supplemental Fig. 4B,C). In addition to the first used the Xenopus laevis embryo as a model system endodermal expression, we found Wnt5a to be abundant in and exposed anterior endodermal cells, which are fated the surrounding mesenchyme at both E8.5 and E10.5, to become either liver or pancreas, to soluble Wnt5a pro- suggesting potential autocrine and/or paracrine ligand tein. In addition to the expected morphogenetic defects, activities (Supplemental Fig. 4C). The other noncanonical Wnt5a induced pancreatic progenitor gene expression, Wnts, Wnt5b and Wnt7b, were found to be abundant in including Pdx1 and Ptf1a, and, to a lesser extent, pancre- pancreatic progenitor cells and not in hepatoblasts starting atic differentiation markers, such as Glucagon (Gcg) and from E10.5, while they were expressed at low levels in Trypsin (Fig. 4A,B). Concomitantly, Wnt5a treatment re- foregut cells (Supplemental Fig. 4A,B). pressed expression of the hepatic genes Hex, For1, and Perturbations of Wnt5a signaling in Xenopus, zebra- Albumin in the same endodermal cells without affecting fish, and mice strongly suggest that Wnt5a activates a other endodermal cell fates, as judged by unchanged conserved pathway that controls cell fate, movements, Nkx6.2 (duodenum) and Sox2 (stomach) expression levels and polarity during development (Moon et al. 1993; Ho (Fig. 4B; Chalmers et al. 2000; Dichmann and Harland

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Figure 3. Analysis of candidate regulators of the pancreatic versus hepatic fate decision. (A) Heat map view of the FP group transcripts that were differentially expressed between any of two samples (150 transcripts out of 517; Cufflinks, P-value < 0.05). Colors represent high (red) or low (blue) expression values based on Z-score normalized FPKM values for each gene. White represents the average between red (high) and blue (low) expression values. Dashed boxes highlight gene sets validated by either RT-qPCR or immunofluorescence analyses. (B) RT-qPCR validation of a subset of differentially expressed genes of the FP group. Data were normalized to that of succinate dehydrogenase (SDHA) and are represented as fold change compared with the E8.5 foregut sample (set to 1 as calibrator). Error bars represent 6SEM. (C) Immunofluorescence analysis validated the exclusive localization of Celrs2, Claudin 4 (Cldn4), CK19, Fat1, Fzd2, and Ror2 in E8.5 foregut endoderm (see arrows in the insets) and pancreatic progenitors and their absence in the liver (see arrows). Micrographs show cross-sections of E8.5 and E10.5 mouse embryos. Bars, 50 mm. (duo) Duodenum; (hep) hepatic progenitors; (lv) liver; (nf) neural folds. Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

Rodrı´guez-Seguel et al.

Figure 4. Noncanonical Wnt5a activity promotes pancreatic versus hepatic fate in the anterior endoderm. (A) Xenopus embryos injected with Wnt5a mRNA showed a shortened and mildly bent body at the tailbud stage, as previously described (Kim et al. 2005). (B) Endodermal explants were cultured in the presence of Wnt5a recombinant protein from stage 10, collected at the tadpole stage, and assayed for expression of the indicated pancreatic, hepatic, and duodenum/stomach genes by RT-qPCR analysis. Untreated anterior endodermal explants were used as control. Data were normalized to that of ornithine decarboxylase (ODC) and are represented as fold changes compared with untreated endoderm sample (set to 1 as calibrator). Error bars represent 6SEM. (C) Whole-mount double in situ hybridization analysis of Hex (light blue) and Ptf1a (purple) in control and Wnt5a-injected Xenopus embryos at the tadpole stage. The arrow indicates Hex expression in the liver bud, and arrowheads indicate Ptf1a expression in the two pancreatic buds (dorsal and ventral buds). Dashed lines mark expanded Ptf1a expression in the injected embryos. Total number of injected embryos = 61; 41% showed visible expansion of Ptf1a. (AE) Anterior endoderm; (PE) posterior endoderm. (D) RT-qPCR analysis of endodermal explants treated with Wnt5b (200 ng/mL) recombinant protein. Data were normalized to that of ODC and are represented as fold changes compared with untreated endoderm sample (set to 1 as calibrator). (E) RT-qPCR analysis of endodermal explants treated with 500 ng/mL Wnt3a recombinant protein. Data were normalized to that of ODC and are represented as fold changes compared with untreated endoderm sample (set to 1 as calibrator). (F) RT-qPCR analysis of direct downstream target genes of the Wnt/b-catenin pathway in endodermal explants treated with 500 ng/mL Wnt5a or 500 ng/mL Wnt3a recombinant protein. Data were normalized to that of ODC and are represented as fold changes compared with untreated endoderm sample (set to 1 as calibrator). (G) TOPFLASH and ATF2-luc reporter assays in Xenopus embryos. Four-cell stage embryos were injected into the vegetal blastomeres with 50 pg of TOPFLASH or 100 pg of ATF2-luc plus 25 pg of Renilla luciferase reporter plasmids. Endodermal explants were dissected at stage 9 and either left untreated as control (CTRL) or exposed to 500 ng/mL Wnt5a or 500 ng/mL Wnt3a recombinant protein, as indicated. Luciferase reporter assays were carried out in explants lysed at gastrula and early tailbud stages. (H) Western blot analysis of dissected anterior endodermal explants either left untreated as control (CTRL) or exposed to Wnt5a or Wnt3a recombinant protein. The relative ABC/tubulin levels in the treated explants compared with the control, which was set to 1.0, are indicated. (b-cat) Total b-catenin; (tub) a-tubulin. (*) P < 0.05; (**) P < 0.01, as determined by the REST program statistical analysis (Pfaffl et al. 2002).

2011). Similar effects were observed when Wnt5a mRNA panded Ptf1a expression beyond its normal boundaries was microinjected into either anterior or posterior endo- (Fig. 4C). Furthermore, we found that activation of the dermal cells of the Xenopus embryo, resulting in ex- noncanonical Wnt pathway using a different noncanonical

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RNA-seq of hepatic and pancreatic progenitor cells ligand, the Wnt5b recombinant protein, also induced a Next, we conducted biochemical assays on endoderm strong expression of pancreatic progenitor transcription explants treated with Wnt5a and Wnt3a to analyze the factors in anterior endodermal cells (Fig. 4D). Finally, we status of activation of b-catenin signaling. Western blot examined whether the exposure to the noncanonical Wnt analysis showed that levels of total and active b-catenin pathway has an effect on cell proliferation in the anterior (ABC) protein (dephosphorylated on Ser37 and Thr41) endoderm. Immunostaining of phospho-histone H3 (PHH3) (van Noort et al. 2002) remained unchanged in Wnt5a- to mark cells undergoing mitosis revealed no significant treated anterior endodermal cells when compared with differences in proliferating cell number in the foregut control endoderm (Fig. 4H), whereas Wnt3a moderately between Wnt5a-injected and uninjected early tadpole stage increased dephosphorylated b-catenin levels. As ex- embryos (Supplemental Fig. 4E). pected, Wnt5a induced JNK phosphorylation, reflecting On the other hand, the establishment of proper levels noncanonical Wnt activation (Fig. 4H). Taken together, of canonical Wnt/b-catenin signaling and its temporal these results rule out Wnt5a function as an antagonist sequential activation in the anterior endoderm (Fig. 4) of the canonical Wnt pathway and support a role of the are known to be essential for foregut identity and organ noncanonical Wnt signaling in the pancreatic versus formation (Ober et al. 2006; Li et al. 2008; Angers and hepatic fate decision in a b-catenin-independent manner. Moon 2009; Puri and Hebrok 2010; Zhang et al. 2013). Last, we sought to expand the functional analysis on After adding Wnt3a recombinant protein, a well-known the noncanonical Wnt pathway directly to mammalian canonical Wnt activator (Grigoryan et al. 2008; Clevers endodermal cells. First, we used the mouse embryonic and Nusse 2012), to anterior endodermal cells, we ob- stem cell (mESC) system to model ex vivo endoderm served that the levels of expression of hepatic and pancre- development (Rossant 2011). In particular, to induce atic genes were unchanged when compared with control pancreatic specification, mESCs were stimulated using endoderm, ruling out an apparent choice of cell type a step-wise protocol in a monolayer culture adapted from between liver or pancreas (Fig. 4E). previously published studies (D’Amour et al. 2006; The noncanonical Wnt signaling is able to antagonize Nostro et al. 2011; Wang et al. 2011; Chen et al. 2013). the canonical signaling in certain biological contexts RT-qPCR analysis revealed first up-regulation of defini- (Grumolato et al. 2010; Ho et al. 2012). To distinguish tive endoderm transcription factors such as Sox17 and between these two possible Wnt5a activities (e.g., non- Foxa2 followed by sequential induction of pancreatic canonical Wnt activity or antagonist effect on Wnt/ progenitor genes, including Pdx1, Sox9, Pax6, and Islet1 b-catenin) in the context of liver and pancreas lineage (Isl1) (Fig. 5A). Consistent with pancreatic differentiation, divergence, we performed a series of quantitative assays the levels of expression of Sox9, Pax6, and Isl1 increased and biochemical analysis in the Xenopus endoderm. First, with time and upon exposure to additional cytokines we analyzed the transcriptional responses to both path- (e.g., noggin, retinoic acid, and cyclopamine), while no or ways in endoderm cells treated with Wnt5a by (1) exam- minimal induction of the hepatic marker Albumin was ining direct downstream target genes of Wnt/b-catenin detected (Fig. 5A). Most differentiation protocols use pathway and (2) using specific luciferase reporter assays FGF10 to pattern the definitive endoderm-induced pop- based on TCF/LEF (TOPFLASH) and ATF2 response ulation toward the pancreatic endoderm fate within the elements for canonical and noncanonical Wnt, respec- foregut and the subsequent pancreatic progenitor fate tively (Ohkawara and Niehrs 2011; Clevers and Nusse (D’Amour et al. 2006; Nostro et al. 2011). To determine 2012). RT-qPCR analysis showed that Wnt3a treatment whether the noncanonical Wnt pathway would enhance increased the expression levels of direct Wnt target genes pancreatic specification, we examined the consequences such as Cyclin-D1 (Ccdn1), Lef-1, Myc,andAxin 2, of treating definitive endoderm cultures generated from whereas Wnt5a treatment does not affect their expres- ESCs with Wnt5a in the presence or absence of FGF10. sion in anterior endodermal cells (Fig. 4F). This is in line Interestingly, Wnt5a enhanced the levels of Pdx1 expres- with the RNA-seq profiles of mammalian pancreatic and sion compared with the standard pancreatic endoderm hepatic progenitor cells displaying comparable expres- differentiation conditions (Fig. 5B). In addition, the posi- sion levels of these downstream target genes (Fig. 2; tive effect of Wnt5a was even more evident in definitive Supplemental Table 2). Furthermore, we found that endoderm cells cultured in the absence of FGF10, as its exposure to Wnt5a does not suppress endogenous ca- addition restored the induction of Pdx1 expression to nonical Wnt activity in the endoderm, as monitored by pancreatic endoderm standard levels (Fig. 5C). Impor- the Wnt/b-catenin TOPFLASH reporter assay, whereas tantly, the duodenal marker Cdx2 was not induced in it induces the noncanonical Wnt ATF2 reporter activity pancreatic endoderm cultures differentiated in the pres- (Fig. 4G). Notably, endogenous canonical Wnt/b-catenin ence of Wnt5a, and the slight induction of Albumin transcriptional activity accumulates in the Xenopus endo- expression in pancreatic endoderm cultures was down- derm from gastrulation onward (Fig. 4G; Angers and Moon regulated by Wnt5a treatment, supporting a specific ef- 2009; Ohkawara and Niehrs 2011). This results in elevated fect on pancreatic fate (Fig. 5B). In addition, we found that basal levels of TOPFLASH luciferase activity in the other pancreatic progenitor transcription factors, includ- untreated endodermal cells (control [CTRL]) and is ing Nkx6.1 (at the pancreatic endoderm stage), Sox9, and consistent with the mild induction of the TOPFLASH Pax6 (at the pancreatic progenitor stage), were strongly reporter observed upon exposure to Wnt3a-soluble pro- induced in both the absence and presence of Wnt5a, tein (Fig. 4G). although a further enhancement of their transcript levels

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Figure 5. Conserved Wnt5a activity in promoting pancreatic fate. (A) Directed differentiation of mESC monolayer cultures into pancreatic progenitors. RT-qPCR analysis evaluating definitive endoderm (DE), pancreatic endoderm (PE), and pancreatic progenitor (PP) gene expression at different stages of differentiation. Untreated mESCs were used as control (d0). Data were normalized to that of SDHA and are represented as fold changes compared with control (d0) mESCs (set to 1 as calibrator). (B) Day 5 and day 8 mESC cultures were analyzed by RT-qPCR for the expression of the indicated genes following either standard pancreatic endoderm and pancreatic progenitor culture conditions or in the presence of Wnt5a recombinant protein (PE + Wnt5a and PP + Wnt5a). RT-qPCR data were normalized to that of SDHA and are represented as fold changes compared with control (d0) mESCs (set to 1 as calibrator). Error bars represent 6SEM. (C) Day 5 mESC cultures were analyzed by RT-qPCR for the expression of the pancreatic gene Pdx1 following standard pancreatic endoderm culture conditions in the absence of FGF10 (PE À FGF10), the presence of Wnt5a (PE + Wnt5a), or the presence of Wnt5a but without FGF10 (PE À FGF10 + Wnt5a). RT-qPCR data were normalized to that of SDHA and are represented as fold changes compared with standard pancreatic endoderm condition (set to 1 as calibrator). Error bars represent 6SEM. (D) BAML liver cells cultured in the presence of 200 ng/mL Wnt5a for 2 wk were assayed for expression of the indicated pancreatic and hepatic genes by RT-qPCR analysis. Untreated BMAL liver cells were used as control. Data were normalized to that of SDHA and are represented as fold changes compared with untreated liver cells (set to 1 as calibrator). Error bars represent 6SEM. (*) P < 0.05; (**) P < 0.01, as determined by the REST program statistical analysis (Pfaffl et al. 2002). (Alb) Albumin.

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(as seen for Pdx1) was not detectable in these culture establish the exact contribution of progenitor domains conditions (Fig. 5B). This finding suggests that patterning within the endoderm. of anterior endoderm with noncanonical Wnt signaling Notably, in the ventral foregut epithelium, we detected might be important for optimal pancreatic specification Hox genes encoded by the HoxD clusters (Hoxd3 and in protocols for directed differentiation of ESCs. Hoxd4) as well as a spliced noncoding RNA that is Next, we asked whether modulation of the noncanon- transcribed antisense to the Hoxb5 and Hoxb6 genes ical Wnt pathway might promote the pancreatic program (0610040B09Rik), which might be relevant for their in differentiated liver cells. To this aim, we used a mam- regulation (Fig. 6A; Supplemental Fig. 6; Dinger et al. malian ex vivo model system, the nontransformed BAML 2008). A clear spatial difference in the expression of Hox hepatic cell line (Fouge`re-Deschatrette et al. 2006). The genes was evident also at later stages in the pairwise BAML cells were established from adult mouse livers and comparison between E10.5 vpa and dpa data sets (Supple- were shown previously to retain hepatic differentiation mental Fig. 7). Hox genes such as Hoxa3, Hoxa4, Hoxa7, hallmarks and repopulate the liver in vivo (Fouge`re- Hoxb3, Hoxb6, Hoxb7,andHoxb8 were abundantly Deschatrette et al. 2006). We cultured the liver cells in expressed in the dorsal pancreatic region but were absent their standard hepatocyte culture medium in the pres- or at undetectable levels in the vpa and liver (Fig. 6A; ence or absence of Wnt5a. After prolonged exposure to Supplemental Fig. 7). Using RT-qPCR and in situ hybrid- Wnt5a, we observed significant induction of the pancre- ization, we confirmed the differential expression between atic genes, including Pdx1, Pax6, and MafA, in liver cells, the dpa and vpa of selected Hox genes, including Hoxa7, whereas the level of expression of the liver markers Hoxb6, and Hoxb8 (Fig. 6F–I). Notably, Hoxa7 and Hoxb6 Albumin and a1-antitrypsin (A1AT) and the liver-specific seem to be expressed in a heterogenous manner in the transcription factor HNF4a1 was not affected (Fig. 5C). dpa, possibly marking specific cell types (Fig. 6F,G). This suggests that Wnt5a might also facilitate fate con- Taken together, our findings suggest that a specific Hox version of liver cells into pancreatic fate but does not code exists in the developing foregut as well as within the itself possess the ability to suppress hepatic identity. pancreatic territory, and its biological significance needs All together, these results implicate a novel and con- to be assessed (Grapin-Botton and Melton 2000; Iimura served role for the noncanonical Wnt signaling pathway et al. 2009). in promoting the pancreas versus liver fate decision in the Besides the Hox genes, we detected large differences in endoderm. In particular, Wnt5a appears to exert specific gene expression between the ventral and dorsal pancreatic regulation on Pdx1 gene expression, which is conserved buds at the onset of pancreas organogenesis (192 tran- in both the mouse and Xenopus endoderm (Figs. 4, 5). An scripts; absolute value of log2 fold change > 3, P-value < entire set of noncanonical Wnt transducers was identified 0.05) (Supplemental Fig. 7). To determine whether there in our analysis (Figs. 2, 3); whether Wnt5a and Wnt5b was any recognizable biological relevance to the expression activities in this cell lineage decision are due to signaling patterns, we analyzed GO terms with respect to molecular through components of the PCP, Ror2/JNK, or Calcium/ functions and found significantly enriched annotations in NFAT pathways (Kim et al. 2005; Ober et al. 2006; Li each cluster. Strong association with extracellular bind- et al. 2008; Puri and Hebrok 2010) deserves further ing activities (GO: 0005488; 62 genes; P-value < 0.000129) investigation. was found in the vpa cluster, whereas the dpa cluster showed strong enrichment for DNA-binding transcription factor function (GO: 0003700; 25 genes; P-value < Spatially distinct expression patterns mark 9.41310À18). the progenitor populations Our comprehensive analysis further illustrates how Our data provide an opportunity to systematically exam- different the dorsal and ventral pancreatic progenitors ine additional in vivo expression patterns within the are from each other. Despite these differences at E10.5, hepatic and pancreatic endoderm. For example, we fo- both pancreatic rudiments give rise to endocrine and cused on identifying transcriptional differences between exocrine cells at later stages and eventually fuse to form discrete domains of the foregut endoderm at E8.5 (fg vs. the definitive pancreas (Puri and Hebrok 2010). Our data mfg), before organ rudiments were formed (Fig. 6). Only set will enable a clear understanding of how distinct 21 transcripts displayed statistically significant differen- transcriptional programs might lead to the same cell type, tial expression between these two regions (Fig. 6A). allowing, for instance, the identification of alternative The small number of differentially expressed transcripts modes that can be used to program stem cells toward might reflect the active movements and intermingling of pancreatic endocrine fates. cells across the foregut at this stage (Tremblay and Zaret 2005). Among them were only a few genes that had been Concordant gene signatures between mouse previously reported in the endoderm, including Pyy, Otx2, and human pancreatic progenitors and Sox2 (Hou et al. 2007), but the majority was not known to be expressed in this territory (Fig. 6A–D). The Next, we sought to evaluate the evolutionary impor- expression of some of these foregut genes was maintained tance of our transcriptome analysis of pancreatic pro- in either liver progenitors (E2f2), ventral pancreatic progenitor cells and its relevance to human pancreas de- genitors (Celsr2 and Nr1h5), or both (Cdkn1c), represent- velopment. Given the fact that genomic analysis on ing interesting candidates for lineage tracing studies to equivalent developmental stage human material is not

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Figure 6. Identification of distinct spatial patterns within the mouse foregut endoderm. (A) Heat map view of the transcripts that showed significant differential expression between E8.5 fg and mfg (21 transcripts). Colors represent high (red), low (blue), or average (white) expression values based on Z-score-normalized FPKM values for each gene. (B) RT-qPCR validation of a subset of the foregut differentially expressed genes. The XLOC_019271 is not supported by any spliced ESTs or Genscan predictions. By exon junction analysis, we predicted a gene model and validated the expression of this novel transcript in the fg and dpa by RT-qPCR (see also Fig. 3B; Supplemental Fig. 8). Data were normalized to that of SDHA and are represented as fold change compared with the E8.5 fg sample (set to 1 as calibrator). Error bars represent 6SEM. (C–H) Immunofluorescence and in situ hybridization analyses validated the expression of the indicated genes in the E8.5 foregut endoderm (see arrowheads) and/or dorsal pancreatic rudiments (demarcated by dashed line). At E8.5, Otx2 expression was detected in the mfg (C), which coexpressed Sox17 and E-cadherin (Ecad), as shown by immunofluorescence staining on serial section (C9). FoxD3 expression was detected in the E8.5 fg endoderm (D; see arrowheads) and dpa cells at E10.5 (E). Arrows in E indicate FoxD3/Pdx1-double-positive cells. At E10.5, Hox gene expression was detected in the dorsal pancreatic rudiment (F–H), which coexpressed Pdx1 and E-cadherin (Ecad), as shown by immunofluorescence staining on serial section (F9). Bars, 50 mm. (I) RT-qPCR validation of the indicated Hox genes showed differential expression between the vpa and dpa. Data were normalized to that of SDHA and are shown as expression ratio (2-log values) of dpa sample versus vpa sample. Error bars represent 6SEM. (J) The heat map shows the expression of 100 mouse genes found to be expressed in both the vpa and dpa (>10 FPKM) and whose human orthologs are also expressed in human pancreatic ‘‘progenitor-like’’ cells (Micallef et al. 2012).

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RNA-seq of hepatic and pancreatic progenitor cells available, we compared our murine RNA-seq data sets Materials and methods with available microarray data obtained from human ESC (hESC)-derived pancreatic-like cells (Micallef et al. Mouse embryo dissection and FACS sorting 2012). The transgenic mouse line Tg(Prox1-EGFP)Gsat/Mmcd was ob- Human pancreatic progenitor state was defined by the tained from the Heintz Laboratory-Gensat Project and generated top 400 up-regulated genes in hESC-derived pancreatic as previously described (Gong et al. 2003). E8.5 and E10.5 Prox1- cells versus undifferentiated hESCs (Micallef et al. 2012). EGFP-positive embryos were selected and dissected using an Importantly, we restricted our analysis only to those epifluorescence stereomicroscope (Discovery V12, Zeiss). Eye- genes that were up-regulated in hESCs differentiated into brow knives or tweezers were used for cutting the GFP-positive a specific pancreatic differentiation stage that precedes embryonic regions. The dissected embryonic regions were disso- the onset of overt b-cell differentiation, representing ciated by trypsin (trypsin/EDTA 0.25% solution) digestion into single-cell suspension. To stop the reaction, DMEM (Invitrogen) the closest gene signature of human pancreatic progeni- was added to the cell suspensions and centrifuged at 300g at 4°C, tors. This corresponds to the fraction of ‘‘FACS-sorted and pellets were suspended and PBS/DEPC-treated. Before sort- insulin-GFP-negative cells’’ obtained after application of ing, propidium iodide was added to select only live cells, and the published differentiation protocols to undifferentiated cell suspension was filtered through a BD Falcon tube with cell GFP/w INS hESCs (Micallef et al. 2012). Mouse genes from strainer cap (BD 352235). After dead cell exclusion (SSC-A/PI-A), the RNA-seq experiments were matched to their corre- GFP-expressing cells were sorted at 4°C using a FACS Aria III sponding human homologs using the NCBI database flow cytometer (BD Biosciences) using a GFP filter and by setting HomoloGene (http://www.ncbi.nlm.nih.gov/homologene; the gate on the GFP fluorescence intensity. Conditions of sorting see also the Materials and Methods). Importantly, we were as follows: 70-mm nozzle and sheath pressure of 70 psi. found that that 25% of the 400 human homolog genes Sorted cells were collected directly in TRIzol reagent (Invitrogen) for RNA extraction. Cells isolated by FACS were used for RNA are also markers of pancreas progenitors in mice, display- isolation and RNA-seq and subsequently for RT-qPCR valida- ing high gene expression in the E10.5 vpa and dpa data tion. Approximately, a total of 10,000 cells per sample were sets despite the methodological and temporal differ- pooled to isolate 100 ng of high-quality total RNA. All animal ences (Fig. 6J). experimentation was conducted in accordance with the local ethics committee for animal care. Discussion In conclusion, we devised a strategy to purify liver and RNA-seq pancreas progenitors directly from the endoderm of RNA-seq was performed using ;70 ng of total RNA quantified Tg(Prox1-EGFP) reporter mouse embryos. Using RNA- by Agilent RNA 6000 Pico kit (Agilent Technologies). The qual- seq, we profiled the purified progenitor cells and iden- ity of RNA samples prior to library preparation was determined tified transcripts that are differentially expressed across using an Agilent Bioanalzyer, and only samples with high RIN the pancreatic and hepatic endoderm at distinct develop- (RNA integrity number) scores (>8.5) were further processed. Briefly, poly(A) RNA was isolated by two rounds of oligo(dT)25 mental stages. Importantly, we uncovered a unique non- Dynabeads (Invitrogen) purification. Purified poly(A) RNA was canonical Wnt signaling signature in the emergence of fragmented for 3.5 min at 94°C using 53 fragmentation buffer pancreas versus liver from endoderm progenitors that (200 mM Tris-acetate at pH 8.1, 500 mM KOAc, 150 mM MgOA) appears conserved across phylogenetically distant spe- as described previously (Adamidi et al. 2011). The fragmented cies. Our results provide an invaluable resource for RNA was purified by Agencourt RNAClean XP SPRI beads lineage tracing analysis to pinpoint the exact origin of (Agencourt) and converted to first strand cDNA using random the hepato-pancreatic lineage and isolation of transient hexamer primers (Invitrogen) and SuperScript II reverse tran- progenitor cell populations. Moreover, our transcrip- scriptase (Invitrogen) followed by second strand cDNA synthe- tional data lay the foundation for further targeted func- sis with Escherichia coli DNA polymerase I (Invitrogen) and tional studies of developmental regulators of the liver and RNase H (Invitrogen) according to the manufacturer’s intstruc- tions. The 76-nucleotide (nt) paired-end sequencing library was pancreas fate decision that will be relevant to humans. prepared using New England Biolabs Next DNA Library prep- Indeed, when we compared our RNA-seq results with aration kit following the Illumina mRNA-Seq library prepara- those previously obtained using microarray in hESCs tion protocol. The minimum necessary number of cycles of differentiated into pancreatic-like progenitors (Micallef amplification was used to minimize amplification biases (15 et al. 2012), we identified concordant gene signatures cycles with no overamplification observed). The quality of cDNA (e.g., similar markers of pancreatic progenitors: Wls, libraries for sequencing was assessed using Agilent Technologies FLRTs, and Meis). These results suggest direct impli- 2100 Bioanalyzer. The prepared sequencing library was subse- cations of our findings in developing novel strategies to quently sequenced on Illumina HiSeq for 2 3 100 cycles following generate pancreas progenitors and b cells for clinical the standard protocol (Adamidi et al. 2011). transplantation from cellular programming of stem cells or induced pluripotent stem cells (iPSCs) and from Bioinformatics analysis reprogramming of adult hepatic cells. Finally, our Mapped reads from TopHat (version 1.33) were used as input for RNA-seq analysis might be used as an in vivo reference Cufflinks (version 1.3.0) (Trapnell et al. 2010) for transcript for direct comparison with diseased human tissues assembly and differential expression using the University of such as pancreas dysplasia or agenesis or congenital liver California at Santa Cruz mm9 reference annotation. To obtain defects. a working data set for the purposes of comparing transcriptomes

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Rodrı´guez-Seguel et al. between different samples, we filtered the Cufflinks gene set for mined in triplets. All experiments were repeated at least three (1) successful deconvolution of fragments per kilobase of exon times unless otherwise stated. Primer sequences can be obtained per million mapped fragments (FPKM), (2) FPKM > 0 in at least on request. two samples, and (3) lower confidence level of FPKM > 0inat least two samples. In the case that multiple Cufflinks genes for Xenopus embryo experiments one gene symbol existed, Cufflinks genes were further selected Xenopus embryo manipulations and dissections were performed for greatest transcript length and highest variability in the FPKM as described (Spagnoli and Brivanlou 2008). The SP64T-xWnt5a across all samples. This resulted in a nonredundant gene set of plasmid used for microinjection was previously described (Moon 14,053 Cufflinks genes. et al. 1993). For whole endodermal explant experiments, endo- Unless otherwise noted, bioinformatic analysis was carried dermal cells were microdissected at stage 9 and cultured in 0.13 out using R statistical environment (version $2.15; http:// MMR in the presence of the indicated recombinant proteins www.r-project.org). For the PCA of the nonredundant working until the desired stage. For anterior endodermal explants, dorsal data set, a ceiling FPKM value was set as the 90th percentile of vegetal cells were microdissected at stage 10 and cultured as all expression values in the data set. The first two principal above. Ornithine decarboxylase (ODC) was used as a reference components exhibited 48% and 35% of the total variability for gene in Xenopus RT-qPCR analysis. All of the values were PC1 and PC2, respectively. The Venn diagram was generated normalized to the reference gene and calculated using the REST using the R packages limma (Ritchie et al. 2007) and VennDiagram program (Pfaffl et al. 2002). For luciferase reporter assays in (Adamidi et al. 2011). Enriched GO categories were calculated Xenopus, embryos were injected with ATF2-luc (Ohkawara and using the Bioconductor package TopGO (Alexa et al. 2006). In each Niehrs 2011) or TOPFLASH and Renilla-TK plasmid DNA case, the enrichment of terms in the group FP was compared with (Clevers and Nusse 2012). Three pools of 15 explants each were all genes in the working data set with annotations in their re- lysed with passive lysis buffer and assayed for luciferase activity spective ontologies. using the Dual Luciferase system (Promega). Recombinant mouse To demonstrate relevance with human pancreatic develop- Wnt5a, Wnt5b, and Wnt3a (R&D System) proteins were used at ment, we obtained normalized microarray data from a previously the indicated concentrations in both Xenopus and cell culture published study of hESC differentiation to pancreatic cells experiments. All experiments were repeated at least three times (Micallef et al. 2012). Differential gene expression (at a false dis- unless otherwise stated. covery rate [FDR] adjusted P-value < 0.05) between the differentiated pancreas-like cells (‘‘Nostro protocol’’) and hESC lines was Cell culture experiments determined using the R statistical package limma (Smyth 2005). Mouse genes from the RNA-seq experiments were matched to mESCs (R1 mESC line) were maintained on gelatin-coated plates their corresponding human homologs using the NCBI database with mouse embryonic fibroblasts (MEFs) in standard mESC HomoloGene (http://www.ncbi.nlm.nih.gov/homologene). The medium: DMEM (Invitrogen), 2 mM glutamax (Invitrogen), RNA-seq data sets have been deposited in NCBI’s Gene Expres- 1 mM sodium pyruvate (Invitrogen), 0.1 mM nonessential amino sion Omnibus (GEO) and are accessible through accession number acids (Invitrogen), 15% fetal bovine serum (FBS) (PAN Biotech), GSE40823. 0.1 mM b-mercaptoethanol (Sigma), and 1000 U/mL leukemia inhibitory factor (ESGRO). For differentiation, cultures were MEF-depleted and seeded in mESC medium at high confluency Immunohistochemistry and in situ hybridization on gelatin-coated dishes. Monolayer differentiation was carried Mouse embryos were fixed in 4% paraformaldehyde from 2 h to out as described previously (D’Amour et al. 2006; Nostro et al. overnight at 4°C. Subsequently, samples were equilibrated in 2011). Briefly, definitive endoderm medium to day 2 consisted of 20% sucrose solution and embedded in OCT compound (Sakura). RPMI medium (Invitrogen) and 0.2% FBS supplemented with 50 In situ hybridization on cryostat sections was done as in ng/mL Activin A and 25 ng/mL Wnt3a at day 1 and Activin A at Schaeren-Wiemers and Gerfin-Moser (1993). Cryosections (10 day 2. Pancreatic endoderm medium to day 5 consisted of RPMI mm) were incubated with TSA (Perkin Elmer) blocking buffer for medium and 2% FBS supplemented with 3 ng/mL Wnt3a and 1 h at room temperature and afterward with primary antibodies 50 ng/mL FGF10. Pancreatic progenitor medium to day 8 consisted at an appropriate dilution (Supplemental Table 4). All confocal of DMEM with 1% (v/v) B27 supplement (Invitrogen), 50 mg/mL images were acquired with an LSM 700 confocal laser-scanning ascorbic acid (Sigma), 0.25 mM KAAD-cyclopamine (Toronto Re- microscope (Zeiss). search Chemicals), 2 mMall-trans retinoic acid (Sigma), and 50 ng/mL noggin. All recombinant proteins were purchased from R&D System unless otherwise stated. The BAML cells were RT-qPCR cultured as previously described (Fouge`re-Deschatrette et al. 2006). For RNA isolation, embryonic tissues were microdissected, FACS-sorted, and collected in TRIzol reagent (Invitrogen). Sub- Western blot analysis sequently, RNA was extracted from a minimum of 3000 cells For Western blot analysis, endodermal cells were lysed as de- according to the manufacturer’s instructions (Roche). Briefly, scribed (Spagnoli and Brivanlou 2008). Immunoblots were in- total RNA was resuspended in 10 mL of DEPC H O and processed 2 cubated with anti-ABC monoclonal antibody, which recognizes for reverse transcription using Transcriptor First Strand cDNA the dephosphorylated b-catenin on Ser37 and Thr41 (Millipore); Synthesis kit (Roche). A mix of anchored oligo(dT)18 and random anti-b-catenin polyclonal antibody (Santa Cruz Biotechnology); hexamer primers was used to generate the cDNA. Thermolabile anti-pJNK monoclonal antibody (Santa Cruz Biotechnology); nuclease from the Real-Time Ready Cell Lysis kit (Roche) was and anti-a-tubulin monoclonal antibody (Sigma) and analyzed added to the reverse transcription reaction to degrade dsDNA. using the LI-COR Odyssey system. Real-time PCR reactions were carried out using the SYBR Green Master mix (Roche) on the ABI StepOne Plus system. Succinate Statistical tests dehydrogenase (SDHA) was used as reference gene. All of the values were normalized to the reference gene and calculated All results are expressed as mean 6 standard errors. The using the REST program (Pfaffl et al. 2002). Data were deter- significance of differences between groups was evaluated with

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Student’s t-test. P < 0.05 was considered statistically significant. 2006. Plasticity of hepatic cell differentiation: Bipotential Statistical tests relevant to the RNA-seq data set analysis are adult mouse liver clonal cell lines competent to differentiate described in the ‘‘Bioinformatic Analysis’’ section. in vitro and in vivo. Stem Cells 24: 2098–2109. Gong S, Zheng C, Doughty M, Losos K, Didkovsky N, Schambra Acknowledgments U, Nowak N, Joyner A, Leblanc G, Hatten M, et al. 2003. A gene expression atlas of the central nervous system based on We thank W. Birchmeier for discussion and critical reading of the bacterial artificial chromosomes. Nature 425: 917–925. manuscript, and all members of the Spagnoli, Andrade, and Chen Grapin-Botton A, Melton D. 2000. Endoderm development: From laboratories for helpful discussions. We thank the Max Delbru¨ ck patterning to organogenesis. Trends Genet 16: 124–130. Center FACS Core Facility and Dr. Hans-Peter Rahn for support Grigoryan T, Wend P, Klaus A, Birchmeier W. 2008. Deciphering and technical assistance. We are grateful to T. Uemura for the the function of canonical Wnt signals in development and Celsr2 antibody, Y. Minami for the Ror2 antibody, and C. Niehrs disease: Conditional loss- and gain-of-function mutations of for the ATF2-luc plasmid. This research was supported by in- b-catenin in mice. Genes Dev 22: 2308–2341. stitutional funds from the Helmholtz Association and the Grumolato L, Liu G, Mong P, Mudbhary R, Biswas R, Arroyave Bundesministerium fu¨ r Bildung und Forschung (BMBF). F.M.S. is R, Vijayakumar S, Economides A, Aaronson S. 2010. Canon- supported by the European Research Council (ERC)-Starting ical and noncanonical Wnts use a common mechanism to Hepatopancreatic Grant 243045. activate completely unrelated coreceptors. Genes Dev 24: 2517–2530. References Ho H, Susman M, Bikoff J, Ryu Y, Jonas A, Hu L, Kuruvilla R, Greenberg M. 2012. Wnt5a-Ror-Dishevelled signaling con- Adamidi C, Wang Y, Gruen D, Mastrobuoni G, You X, Tolle D, stitutes a core developmental pathway that controls tissue Dodt M, Mackowiak S, Gogol-Doering A, Oenal P, et al. morphogenesis. Proc Natl Acad Sci 109: 4044–4051. 2011. De novo assembly and validation of planaria tran- Hou J, Charters A, Lee S, Zhao Y, Wu M, Jones S, Marra M, scriptome by massive parallel sequencing and shotgun pro- Hoodless P. 2007. A systematic screen for genes expressed in teomics. Genome Res 21: 1193–1200. definitive endoderm by serial analysis of gene expression Alexa A, Rahnenfu¨ hrer J, Lengauer T. 2006. Improved scoring of (SAGE). BMC Dev Biol 7: 92. functional groups from gene expression data by decorrelating Iimura T, Denans N, Pourquie´ O. 2009. Establishment of Hox GO graph structure. Bioinformatics 22: 1600–1607. vertebral identities in the embryonic spine precursors. Curr Angers S, Moon R. 2009. Proximal events in Wnt signal trans- Top Dev Biol 88: 201–234. duction. Nat Rev Mol Cell Biol 10: 468–477. Karner C, Chirumamilla R, Aoki S, Igarashi P, Wallingford J, Burke Z, Oliver G. 2002. Prox1 is an early specific marker for Carroll T. 2009. Wnt9b signaling regulates planar cell polar- the developing liver and pancreas in the mammalian foregut ity and kidney tubule morphogenesis. Nat Genet 41: 793– endoderm. Mech Dev 118: 147–155. 799. Chalmers A, Slack J, Beck C. 2000. Regional gene expression in Kikuchi A, Yamamoto H, Sato A. 2009. Selective activation the epithelia of the Xenopus tadpole gut. Mech Dev 96: 125– mechanisms of Wnt signaling pathways. Trends Cell Biol 19: 128. 119–129. Chen A, Borowiak M, Sherwood R, Kweudjeu A, Melton D. Kim H, Schleiffarth J, Jessurun J, Sumanas S, Petryk A, Lin S, 2013. Functional evaluation of ES cell-derived endodermal Ekker S. 2005. Wnt5 signaling in vertebrate pancreas de- populations reveals differences between Nodal and Activin velopment. BMC Biol 24: 23. A-guided differentiation. Development 140: 675–686. Lee C, Friedman J, Fulmer J, Kaestner K. 2005. The initiation of Chung W, Shin C, Stainier D. 2008. Bmp2 signaling regulates liver development is dependent on Foxa transcription fac- the hepatic versus pancreatic fate decision. Dev Cell 15: 738– tors. Nature 435: 944–947. 748. Li Y, Rankin S, Sinner D, Kenny A, Krieg P, Zorn A. 2008. Sfrp5 Clevers H, Nusse R. 2012. Wnt/b-catenin signaling and disease. coordinates foregut specification and morphogenesis by an- Cell Mol Life Sci 149: 1192–1205. tagonizing both canonical and noncanonical Wnt11 signaling. D’Amour K, Bang A, Susan Eliazer S, Kelly O, Agulnick A, Genes Dev 22: 3050–3063. Smart N, Moorman M, Kroon E, Carpenter M, Baetge E. Micallef S, Li X, Schiesser JV, Hirst CE, Yu QC, Lim SM, Nostro 2006. Production of pancreatic hormone–expressing endo- MC, Elliott DA, Sarangi F, Harrison LC, et al. 2012. INSGFP/w crine cells from human embryonic stem cells. Nat Biotech- human embryonic stem cells facilitate isolation of in nol 24: 1392–1401. vitro derived insulin-producing cells. Diabetologia 55: Deque´ant M, Glynn E, Gaudenz K, Wahl M, Chen J, Mushegian 694–706. A, Pourquie´ O. 2006. A complex oscillating network of sig- Miki R, Yoshida T, Murata K, Oki S, Kume K, Kume S. 2012. naling genes underlies the mouse segmentation clock. Science Fate maps of ventral and dorsal pancreatic progenitor cells 314: 1595–1598. in early somite stage mouse embryos. Mech Dev 128: 597– Deutsch G, Jung J, Zheng M, Lo´ra J, Zaret KS. 2001. A bipotential 609. precursor population for pancreas and liver within the embry- Mitiku N, Baker J. 2007. Genomic analysis of gastrulation and onic endoderm. Development 128: 871–881. organogenesis in the mouse. Dev Cell 13: 897–907. Dichmann D, Harland R. 2011. Nkx6 genes pattern the frog Moon R, Campbell R, Christian J, McGrew L, Shih J, Fraser S. neural plate and Nkx6.1 is necessary for motoneuron axon 1993. Xwnt-5A: A maternal Wnt that affects morphogenetic projection. Dev Biol 349: 378–386. movements after overexpression in embryos of Xenopus Dinger M, Amaral P, Mercer T, Pang K, Bruce S, Gardiner B, laevis. Development 119: 97–111. Askarian-Amiri M, Ru K, Solda` G, Simons C, et al. 2008. Long Nostro M, Sarangi F, Ogawa S, Holtzinger A, Corneo B, Li X, noncoding RNAs in mouse embryonic stem cell pluripotency Micallef S, Park I, Basford C, Wheeler M, et al. 2011. Stage- and differentiation. Genome Res 18: 1433–1445. specific signaling through TGFb family members and WNT Fouge`re-Deschatrette C, Imaizumi-Scherrer T, Strick-Marchand regulates patterning and pancreatic specification of human H, Morosan S, Charneau P, Kremsdorf D, Faust D, Weiss M. pluripotent stem cells. Development 138: 861–871.

GENES & DEVELOPMENT 1945 Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

Rodrı´guez-Seguel et al.

Ober E, Verkade H, Field H, Stainier D. 2006. Mesodermal Watanabe T, Nakagawa K, Ohata S, Kitagawa D, Nishitai G, Seo Wnt2b signalling positively regulates liver specification. J, Tanemura T, Shimizu N, Kishimoto H, Wada T, et al. 2002. Nature 442: 688–691. SEK1/MKK4-mediated SAPK/JNK signaling participates in Ohkawara B, Niehrs C. 2011. An ATF2-based luciferase reporter embryonic hepatoblast proliferation via a pathway different to monitor non-canonical Wnt signaling in Xenopus em- from NF-kB-induced anti-apoptosis. Dev Biol 250: 332–347. bryos. Dev Dyn 240: 188–194. Zaret K. 2008. Genetic programming of liver and pancreas Pfaffl MW, Horgan GW, Dempfle L. 2002. Relative expression progenitors: Lessons for stem-cell differentiation. Nat Rev software tool (REST) for group-wise comparison and statisti- Genet 9: 329–340. cal analysis of relative expression results in real-time PCR. Zhang Z, Rankin S, Zorn A. 2013. Different thresholds of Wnt– Nucleic Acids Res 30: e36. Frizzled 7 signaling coordinate proliferation, morphogenesis Puri S, Hebrok M. 2010. Cellular plasticity within the pan- and fate of endoderm progenitor cells. Dev Biol 378: 1–12. creas—lessons learned from development. Dev Cell 18: 342– 356. Ritchie M, Silver J, Oshlack A, Holmes M, Diyagama D, Holloway A, Smyth G. 2007. A comparison of background correction methods for two-colour microarrays. Bioinformatics 23: 2700– 2707. Rossant J. 2011. The impact of developmental biology on pluripotent stem cell research: Successes and challenges. Dev Cell 21: 20–23. Schaeren-Wiemers N, Gerfin-Moser A. 1993. A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: In situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100: 431–440. Slack JM. 2007. Metaplasia and transdifferentiation: From pure biology to the clinic. Nat Rev Mol Cell Biol 8: 369–378. Smyth GK. 2005. Limma: Linear models for microarray data. In Bioinformatics and computational biology solutions using R and bioconductor (ed. R Gentleman et al.), pp. 397–420. Springer, New York. Spagnoli FM. 2007. From endoderm to pancreas: A multistep journey. Cell Mol Life Sci 64: 2378–2390. Spagnoli FM, Brivanlou AH. 2008. The Gata5 target, TGIF2, defines the pancreatic region by modulating BMP signals within the endoderm. Development 135: 451–461. Trapnell C, Williams B, Pertea G, Mortazavi A, Kwan G, van Baren M, Salzberg S, Wold B, Pachter L. 2010. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28: 511–515. Tremblay KD, Zaret KS. 2005. Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues. Dev Biol 280: 87–99. Tumbar T, Guasch G, Greco V, Blanpain C, Lowry W, Rendl M, Fuchs E. 2004. Defining the epithelial stem cell niche in skin. Science 303: 359–363. van Noort M, Meeldijk J, van der Zee R, Destree O, Clevers H. 2002. Wnt signaling controls the phosphorylation status of b-catenin. J Biol Chem 277: 17901–17905. Vichas A, Zallen J. 2011. Translating cell polarity into tissue elongation. Semin Cell Dev Biol 22: 858–864. Wallingford J. 2012. Planar cell polarity and the developmental control of cell behavior in vertebrate embryos. Annu Rev Cell Dev Biol 28: 627–653. Wandzioch E, Zaret K. 2009. Dynamic signaling network for the specification of embryonic pancreas and liver progenitors. Science 324: 1707–1710. Wang Q, Piotrowska K, Ciemerych M, Milenkovic L, Scott M, Davis R, Zernicka-Goetz M. 2004. A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo. Dev Cell 6: 133–144. Wang P, Rodriguez R, Wang J, Ghodasara A, Kim S. 2011. Targeting SOX17 in human embryonic stem cells creates unique strategies for isolating and analyzing developing endoderm. Cell Stem Cell 8: 335–346.

1946 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

Mutually exclusive signaling signatures define the hepatic and pancreatic progenitor cell lineage divergence

Elisa Rodríguez-Seguel, Nancy Mah, Heike Naumann, et al.

Genes Dev. 2013, 27: Access the most recent version at doi:10.1101/gad.220244.113

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